Chronic spontaneous urticaria: Evidence of systemic microcirculatory changes

Abstract Background Chronic spontaneous urticaria (CSU) is a chronic inflammatory skin disease where activation of endothelial cells (ECs) at sites of skin lesions leads to increased blood flow, leakage of fluid into the skin, cellular infiltration, and vascular remodeling. To understand the disease duration and the sometimes vague systemic symptoms accompanying flares, the objective of this study was to examine if CSU comes with systemic vascular changes at the microcirculatory level. Methods We investigated CSU patients (n = 49) and healthy controls (HCs, n = 44) for microcirculatory differences by nailfold videocapillaroscopy (NVC) and for blood levels of the soluble EC biomarkers serum vascular endothelial growth factor (VEGF), soluble E‐selectin, and stem cell factor (SCF). Patients were also assessed for clinical characteristics, disease activity, and markers of autoimmune CSU (aiCSU). Results CSU patients had significantly lower capillary density, more capillary malformations, and more irregular capillary dilations than HCs on NVC. Serum levels of VEGF, soluble E selectin and SCF were similar in CSU patients and HCs. CSU patients with higher VEGF levels had significantly more abnormal capillaries. Patients with markers of aiCSU, that is, low IgE levels or increased anti‐TPO levels, had significantly more capillaries and less capillary dilations than those without. Conclusion Our results suggest that CSU comes with systemic microcirculatory changes, which may be driven, in part, by VEGF.

In CSU, the activation of skin ECs in response to MC degranulation involves the effects of histamine and other mediators (which may include Vascular Endothelial Growth Factor [VEGF]) 11 on H1 receptors. 12VEGF is a potent regulator of angiogenesis, and its expression is abundant in several chronic inflammatory diseases.
VEGF induces proliferation, migration and tube formation of ECs and promotes the expression of chemokines as well as leukocyte adhesion molecules, such as E-selectin.VEGF is produced and secreted by MCs including human skin MCs, 13,14 and the serum of patients with CSU can induce MCs to secrete VEGF in vitro. 15Also, increased blood and lesional skin levels of VEGF have been reported in CSU patients, [16][17][18] although this has not been confirmed. 19,202][23] It plays an important role in transendothelial cell migration, 6,24 and is upregulated in non-lesional and lesional skin of urticaria patients. 11ere is also evidence of vascular remodeling in CSU skin, with significantly more CD31þ ECs in lesional versus non-lesional skin. 25tients with CSU with high numbers of cutaneous CD31þ ECs have high rates of recurrent AE. 26 Not only the formation of new blood vessels, but also the formation of blood cells (hematopoiesis), f.e. through Stem Cell Factor (SCF), has been examined in CSU.Activated ECs release SCF, which is commonly known as the ligand for c-KIT receptor, the main growth factor of MCs. 27,28Nevertheless, the role of SCF in CSU pathogenesis remains insufficiently understood. 29,30nce the integrity of vessel walls is compromised in recurring CSU wheals, we examined if there are changes in the microcirculation outside of the lesional skin area.Therefore, the objectives of this study were to firstly identify whether systemic vascular changes in CSU occur, and secondly, to detect any differences between CSU endotypes (clinically through NVC and biologically through the assessment of specific soluble EC biomarkers [VEGF, sE-selectin, and SCF]).
(CHU) Brugmann, Université Libre de Bruxelles (ULB), Brussels, Belgium, between October 2018 and September 2021.Most patients (67%) were female, the average age was 38.6 � 2.1 years, and 73% were <45 years old.All patients had expert-confirmed CSU, with recurrent wheals (hives) with or without AE for longer than 6 weeks. 31Patients with standalone chronic inducible urticaria, AE only, or wheals due to other causes, that is, infections or food/drug cross-intolerance, were excluded based on clinical history and appropriate investigations.Inclusion criteria consisted of: (a) age >17 years, (b) no antihistamine intake during the 7 days prior to inclusion, (c) not using omalizumab, corticosteroid drugs or cyclosporin treatment at the time of inclusion, (d) no anti-inflammatory drugs at the time of inclusion, and (e) no history of systemic connective tissue disorders.Study patients were assessed for CSU symptom onset, frequency and activity level, diurnal variation, general CSU symptoms and accompanying gastro-intestinal complaints, CSU triggers, previous CSU treatment, occurrence and characteristics of AE, cardiovascular profile, and history of mental health issues.Also, presence of stress, personal and familial medical history were documented, based on the subjective assessment given by the patient.
Furthermore, 44 age-and sex-matched healthy controls (HCs) were included in the study (64% female, mean age:

| Patient-reported outcome measures and comorbidity risk stratification tools
CSU disease activity was determined using the weekly Urticaria Activity Score (UAS7) and defined as: urticaria-free (0 points); well-controlled/minimal disease activity (1-6 points); mild disease activity (7-15 points); moderate disease activity (16-27 points); and severe disease activity (28-42 points). 32SCORE2 (Systematic Coronary Risk Evaluation) and SCORE2-Older Persons (SCORE2-OP; for patients aged >69 years) risk prediction algorithms were used to estimate if the 10-year risk of cardiovascular events was low (Grade 1), moderate (Grade 2), or high (Grade 3). 33,34Patients were categorized as obese when their body mass index score was ≥30 kg/m 2 . 35tients with CSU were assessed for markers of type IIb aiCSU, that is, history or presence of AID, antinuclear antibodies (ANAs) and low IgE levels and elevated anti-TPO levels.As for the latter two, patients were categorized into two groups: (a) patients with low IgE levels, that is, <40 IU/mL, and/or increased anti-TPO levels (n = 19) 36

| Autologous serum skin tests
The autologous serum skin test, ASST is a test for autoreactivity that evaluates the presence of serum histamine-releasing factors including histamine-releasing autoantibodies.The ASST was performed according to the European Academy of Allergy & Clinical Immunology/GA 2 LEN task force consensus procedure as previously described. 37Wheal responses were measured at 15 and 30 min after the intradermal injection of 50 μL of autologous serum.When a red serum-induced wheal had a diameter of at least 1.5 mm greater than the negative control, the ASST response was considered positive.

| Blood analysis
Venous blood was collected from patients with CSU (on the same day as the NVC imaging) and from a part of the HC group (due to logistical reasons, the HCs from UZ Ghent did not participate in this sub study).VEGF, E-selectin, and SCF plasma concentrations were determined using enzyme-linked immunosorbent assay (ELISA), in accordance with the manufacturer's instructions (R&D Systems Inc., Minneapolis, MN, USA).

| Nailfold videocapillaroscopy
CSU and HC study subjects were also investigated by standardized NVC as described by Smith et al 7 .All fingers, except for the thumbs, were assessed with a x200 magnification contact lens (Optilia microscope).Two images per nailfold/finger were captured, coded, and saved (sets of 16 images per patient).
The NVC images were randomized, read, and reported by a trained European League Against Rheumatism (EULAR)-certified capillaroscopic expert (YM) blinded to the subject's health or disease status.We followed the capillaroscopic protocol from the EULAR study group on microcirculation in Rheumatic Diseases (SG MC/RD) definitions that is, the quantitative NVC-assessment in a 1 mm grid 8,38 :

Capillary density
The mean number of capillaries in the distal row per linear mm

Capillary dimension
The mean number of dilated capillaries (dilations) when the apical limb diameter was 20-50 μm and the mean number of giant capillaries when the apical limb diameter was >50 μm; where regular dilations were defined as homogeneously enlarged capillaries and irregular dilations were considered when a segment of the vessel was more dilated than the rest; with the number of dilations, relative to the total number of capillaries per linear mm, represented as percentage, that is, relative capillary dimension (Continues) MOSTMANS ET AL. (Continued)

Capillary morphology
The mean number of capillaries with a normal morphology: Capillaries with a hairpin shape, once or twice-crossing shape, tortuous shape that is, limbs bend but not crossed; on the condition that the tip of the capillary is convex Abnormal morphology: All capillaries whose shape does not correspond to the definition of a normal shape, subcategorized as ramified, concave tip or > twice-crossing shape

| Demographics, clinical characteristics, and laboratory results of patients with chronic spontaneous urticaria
Most patients (70%) had moderate or severe disease activity, based on UAS7 values, 80% had wheals every day, and 49% had CSU for more than 1 year (Table 1).Across all patients, 71% reported more night-time than daytime whealing.AE occurred in 67% of patients, and 78% with AE had attacks at least once per month.In 67% of patients with AE, the lower lip was affected, and 9% reported genital AE.One in five patients (22%), previously or currently, had an autoimmune comorbidity, most commonly Hashimoto's thyroiditis (36%).
One in three patients (35%) had elevated C-reactive protein levels, and one in four (26%) had increased erythrocyte sedimentation rate levels (Table 2).IgE levels were low (<40 IU/ml) and high (>100 IU/ml) in 25% and 50% of patients, respectively, and one fifth of patients (19%) had circulating anti-TPO antibodies.IgE levels were low and/or anti-TPO levels were high in 19/49 (39%) patients.This patient group did not significantly differ in disease duration nor disease activity from patients with high/normal IgE levels and/or normal anti-TPO levels (p = 0.51 and p = 0.88 respectively).
In contrast, patients with CSU and HCs did not differ in the presence of microhemorrhages (p = 0.38) and subpapillary venous plexus visibility scores (p = 0.67).).In contrast, low IgE levels or elevated anti-TPO alone, elevated levels of ANA, and positive ASST results were not linked to significant differences in NVC parameters (Table 4).

| Vascular endothelial growth factor, sE-selectin and history of autoimmune disease were associated with changes in the microcirculation
VEGF and sE-selectin blood levels in patients with CSU, were linked to microcirculatory changes detected by NVC, albeit similar to the levels in HCs (Table 5).Patients with high VEGF levels had signifi-  7,38 .NVC parameters were corrected for age and sex.Capillary crossings were considered abnormal when >2 crossings were observed.The total number of abnormal shapes corresponded with the sum of all capillaries with multiple crossings (>2), ramifications and concave tips.
patients with non-aiCSU.In our study, which was also done at a specialist urticaria center, patients with markers of aiCSU did not have significantly different disease durations nor disease activity compared with patients without markers of aiCSU.In any case, our results suggest that NVC could be a useful clinical tool for the identification of aiCSU patients.
We assessed possible mechanisms of microcirculatory changes on NVC in patients with CSU by looking at certain mediators, that is, sEselectin, SCF and VEGF, all of which are known to activate ECs and regulate vascular permeability.Although many symptoms of urticaria are mediated primarily by the actions of histamine on H1 receptors on ECs (resulting in wheals), 12 other histamine-independent mechanisms appear to contribute to vascular leakage, with direct or indirect influence on VEGF signaling. 11In CSU skin, VEGF is a potential marker for angiogenesis 17,25 ; nevertheless, for CSU serum clear evidence is still lacking. 17,19,20Although there is increasing evidence that VEGF is overexpressed in different conditions including chronic inflammation, we were unable to demonstrate significantly upregulated VEGF levels in the blood of patients with CSU compared to HCs.Nevertheless, patients with CSU with increased VEGF levels had significantly more abnormal capillaries and, more crossing capillaries compared with those with lower VEGF levels.6][47][48][49][50] For systemic sclerosis or HAE-C1INH, no NVC correlations with VEGF have been found. 40,51,52Although several authors show upregulation of E-selectin in (lesional and non-lesional) skin of patients with CSU, [53][54][55][56][57] research on sE-selectin serum levels is controversial 55,58 and lacking. 11In our study, sE-selectin serum levels were not significantly elevated compared with HCs.Furthermore, we were not able to show elevated serum SCF levels in CSU, which is in line with earlier studies of serum SCF in CSU and chronic inducible urticaria. 30r study has several strengths.We performed comprehensive nailfold capillary analyses by NVC, a golden standard approach for the identification and characterization of systemic microcirculatory changes.Moreover, our patients were well characterized clinically and in terms of laboratory markers.Three possible molecular EC markers of microcirculatory changes were observed in combination with NVC assessment.Our NVC findings underline the potential microvascular impact of the chronic nature of CSU and offer perspectives for new research on why CSU has a long disease duration, recurs frequently after years or sometimes comes with aspecific systemic clinical features.0][61][62] Although the NVC changes found in our study seemingly remain subclinical in patients with CSU, future studies should focus more on the detection and origin of long term systemic (vascular) complaints and comorbidities.
As for limitations, firstly, our study was monocentric, and the number and diversity of our patients were limited, for NVC as well as for ELISA.Also, there is a lack of information on aiCSU-defining outcomes such as MC-activating autoantibodies and basophil testing in our study that does not allow for definite conclusions.
Therefore, further studies are needed and should be performed across several centers with larger and more diverse patient populations.The global network of urticaria centers of reference and excellence appears ideally suited to perform such studies. 63Secondly, though NVC is the golden standard method for detecting microvasculopathy in rheumatic diseases, its validity in inflammatory skin diseases remains poorly investigated challenging the interpretation of our results.Since NVC is readily available, straightforward, and inexpensive, further application of NVC in future CSU studies should be combined with clinical and serological assessments.

| CONCLUSION
Taken together, our study identifies abnormalities of the microcirculation in CSU patients and provides further evidence that CSU has systemic effects beyond the development of skin lesions.Circulating
and (b) patients with high/normal IgE levels and normal anti-TPO levels (n = 29).Furthermore, NVC parameters were compared in patients with low IgE values versus normal/high IgE values and in patients with increased anti-TPO levels versus normal anti-TPO levels.

F I G U R E 1 T A B L E 4
Microvascular abnormalities on NVC in patients with chronic spontaneous urticaria and healthy controls.NVC images of six different CSU patients (A-F) showing (A-B) decreased capillary density, 5 capillaries/1 mm grid and 6 capillaries/1 mm grid respectively, (A-D) irregular dilations (>20 μm) (þ), and (B-F) abnormally shaped capillaries including multiple crossing capillaries (*), ramified ("bushy") capillaries (bold arrow) and concave tips (regular arrow).NVC images of 2 different healthy controls (G-H) show a capillary density of >7/mm, with normally shaped hairpin capillary loops and capillary dimensions <20 μm.CSU, chronic spontaneous urticaria; NVC, nailfold videocapillaroscopy.Nailfold videocapillaroscopy findings in CSU patients with aiCSU characteristics compared to CSU patients without.

Maertelaer:
analysis and interpretation of data, critical revising of the article for important intellectual content and final approval of the version to be published and agreed to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.Ines Saidi: acquisition and interpretation of data, critical revising of the article for important intellectual content, final approval of the version to be published and agreed to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.Francis Corazza: acquisition of data, analysis and interpretation of data, critical revising of the article for important intellectual content, final approval of the version to be published and agreed to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.Olivier Michel: substantial contributions to design of the study, acquisition of data, analysis and interpretation of data, drafting of the article, critical revising of the article for important intellectual content, final approval of the version to be published and agreed to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.

reference, mean (SD) CSU (n = 48) HC (n = 20) p-value
NVC images were taken at 200X magnification.At the subject level, the mean of each quantitative parameter was calculated.The averages of the means are reported in this table.Student T-test was used for continuous variables with a cut-off of p < 0.05 for statistical significance (bold).All data are averages � SD for continuous variables.Standard definitions of capillaroscopic evaluations suggested by the European League Against Rheumatism study group on microcirculation in rheumatic diseases (EULAR SG MC/RD) were applied In CSU, sE-selectin levels were linked to a history of AID (65.17 � 6.07 pg/mL, p = 0.002) but not to low IgE and/or high anti-TPO levels (p = 0.278).VEGF and SCF levels in patients with CSU were not linked to a history/presence of AID (p = 0.28 and p = 0.81, respectively) or low IgE and/or high anti-TPO levels (p = 0.299 and p = 0.832, respectively).4|DISCUSSIONaloss of ECs, resulting in reduced capillary density.To further attest to this, longitudinal NVC-studies in CSU are warranted.Interestingly, CSU signature changes of nailfold capillary density, dimensions and morphology were less pronounced in patients with a history/presence of AID and in those with low IgE levels and/or increased anti-TPO levels, which are markers of type IIb T A B L E 1 (Continued) T A B L E 2 Serologic findings of CSU patients and controls.Serologic parameters,Abbreviations: Abs, antibodies; ALP, alkaline phosphatase; ALT, alanine transaminase; ANA, antinuclear antibodies; Anti-TPO, anti-thyroid peroxidase; AST, aspartate aminotransferase; C, complement; CH50, total complement activity; CRP, C-reactive protein; CSU, chronic spontaneous urticaria; ESR, erythrocyte sedimentation rate; fT4, free thyroxine; GGT, gamma-glutamyl transferase; HC, Healthy Controls; H. Pylori, Helicobacter Pylori; IgE, Immunoglobulin E; NA, not applicable; ND, not determined; NS, no significance; TSH, thyroid stimulating hormone.aiCSU.41,42This is unexpected since aiCSU is usually characterized by high disease activity, resistance to standard treatments, and a high rate of AE.43,44At present, we cannot explain why patients with markers of aiCSU show less pronounced capillary abnormalities compared with patients without these markers.In previous studies performed at specialist urticaria centers, patients with markers of aiCSU had a shorter duration of disease, that is, time from the onset of CSU to inclusion in the study.This may be due to the higher disease activity and resistance to standard treatment of aiCSU, driving these patients to seek help faster than T A B L E 3 ELISA results and explorative analysis of quantitative nailfold videocapillaroscopy parameters in CSU patients compared to controls.Demographic data of included population CSU (n = 49) HC (n = 44) Age, n (%) 7,38fold videocapillaroscopy characteristics in CSU patients and their clinico-serological associations.Note: NVC parameters and their clinico-serological associations are given using multiple regressions analyses, including age as a covariate and sex as a factor.p-valuesareconsidered statistically significant when p < 0.05 (bold).Standard definitions of capillaroscopic evaluations suggested by the European League Against Rheumatism study group on microcirculation in rheumatic diseases (EULAR SG MC/RD) were applied7,38.Capillary crossings were considered abnormal when >2 crossings were observed.The total number of abnormal shapes corresponded with the sum of all capillaries with multiple crossings (>2), ramifications and concave tips.
be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.Marcus Maurer: interpretation of data, drafting of the article, critical revising of the article for important intellectual content and final approval of the version to be published and agreed to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.Berappropriately investigated and resolved.Karin Melsens: acquisition and interpretation of data, critical revising of the article for important intellectual content and final approval of the version to be published and agreed to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.Viviane De T A B L E 5Abbreviations: AE, angioedema; AID, auto-immune diseases; ASST, autologous serum skin test; cap, capillaries; CRP, C reactive protein; CSU, chronic spontaneous urticaria; EC, endothelial cell; ESR, erythrocyte sedimentation rate; nb, number; NS, not significant; NVC, nailfold videocapillaroscopy; SCF, stem cell factor; VEGF, vascular endothelial growth factor.